Enzymatically catalyzed reactions pass from reactants to products via transition states that are short-lived and potentially characterized from free-energy reaction surfaces. We compute the reaction surface for Trypanosoma cruzi trans-sialidase using the Free Energy from Adaptive Reaction Coordinate Forces method. The reaction coordinates are the bonds between the sialic acid and the leaving group (TYR342) and the sialic acid and the nucpleophile (ASP59). We are able to track progress of the reaction trajectories up to (incomplete), about (recrossed), and across (crossed) the col that divides the reactant (covalent intermediate) and product (Michaelis complex) surfaces. More than 40 transition state configurations were isolated from these trajectories, and the sialic acid substrate conformations were analyzed as well as the substrate interactions with the nucleophile and catalytic acid/base. A successful barrier crossing requires that the substrate passes through a family of E5, (4)H5, and (6)H5 pucker conformations. These puckers interact slightly differently with the enzyme. The E5 and (4)H5 conformations have a high-frequency hydrogen bonding with Asp96, while (6)H5 puckers show increased hydrogen bonding between sialic acid O-8-Glu230. Our analysis of Trypanosoma cruzi trans-sialidase configurations that populate the col separating the reactant from product surfaces brings new evidence to the prevailing premise that there are several pathways from reactant to product passing through the saddle and successful product formation is not restricted to the minimum energy path and transition state.
The
suppression of competing reactions that lead to side products
is one of the key mechanistic actions defining enzyme catalysis. The
transfer of sialic acid (SA) in a water solution is susceptible to
two competing side reactions, but a single product is the outcome
in the glycosylation and deglycosylation steps of the trans-sialidase (TS) in the Trypanosoma cruzi (T. cruzi) parasite, generally known
as TcTS. We use multidimensional QM/MM free energy computations to
reveal a competition between a minor elimination reaction and the
dominant displacement reaction present in both steps. The simultaneous
monitoring of the progression of the competing reactions reveals lower
barriers in the free energy profiles, a greater sampling of favorable
reactant stereoelectronic alignments, and a greater number of possible
transition paths leading to successful crossing reaction trajectories
for the dominant displacement reactions in comparison with those of
the elimination reactions.
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